Project Summary Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta- hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD is hydroxyurea, which works through upregulating HbF. However, its efficacy is variable among different patients and the mechanism of action is not well understood. Therefore, identifying ways of upregulating HbF, such as inhibiting HbF repressors, is a long-standing interest in this field. BCL11A and LRF are transcription factors that independently function with associated co-regulators to repress HbF, but have limitations in therapeutic potential. While these transcription factors and their co-regulators have been extensively studied, upstream regulation of these transcription factors, such as post-transcriptional regulation, are not well studied. Elucidating these unknown mechanisms may uncover novel therapeutic targets that can bypass the limitations targeting these major HbF repressors hold. To this end, I employed a CRISPR/Cas9 based screening approach to interrogate RNA binding proteins (RBP) in HbF gene regulation. Using HUDEP2 cells, a human erythroid progenitor cell line, we interrogated 527 human RBPs and found that depletion of several RBPs that belong to a category of RBPs termed heterogeneous nuclear ribonucleoproteins (hnRNP) significantly upregulate HbF. Of these proteins, the candidate with the highest effect size was synaptotagmin-binding cytoplasmic RNA interacting protein (SYNCRIP). We validated this result by knocking down SYNCRIP in HUDEP2 cells using CRISPR/Cas9 and assessing the levels of HbF via flow cytometry, western blot, and RT-qPCR. We found that upon SYNCRIP knock down, HbF expression was significantly increased without impacting BCL11A or LRF on the transcriptional and protein level. hnRNP is a category of RBPs that are important for multiple aspects of post transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. Currently, hnRNPs have not been implicated in HbF repression and studies on SYNCRIP in the context of hematopoiesis is limited. I aim to elucidate the mechanisms by which SYNCRIP and other types of hnRNPs work to regulate HbF gene expression. I hypothesize that SYNCRIP, along with other hnRNPs, work to regulate the RNA processing of transcripts encoding co-regulators associated with BCL11A or LRF. In aim 1, I will investigate the role SYNCRIP?s RNA binding activity plays in regulating HbF expression. Notably, two additional hnRNPs known to regulate each other were also identified in this screen. Therefore, in aim 2, I will study the cooperative mechanism of these hnRNPs in HbF regulation. By successfully completing these aims, I will have gained further information on this novel model of HbF repression, which can potentially be exploited for therapeutic purposes in alleviating SCD.